Method for constructing threonine-producing strains

Abstract

The application provides a construction method of a threonine-producing strain. The application applies a pyruvate quinone dehydrogenase inactivated strain (Corynebacterium) to threonine production, and the threonine yield of the strain is increased by 25% to 45.8% compared with that of an unmodified strain. Further combination of the strain with at least one of the expression strengthening phases in aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase and threonine synthase in the threonine synthesis path can increase the threonine yield by 10 to 21 times. The application provides a new way for large-scale production of threonine and has high application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, and more specifically, relates to a method for constructing a threonine-producing strain. Background Technology

[0002] L-Threonine, chemically known as β-hydroxy-α-aminobutyric acid, has the molecular formula C4H9NO3 and a relative molecular mass of 119.12. It is an essential amino acid, primarily used in pharmaceuticals, chemical reagents, food fortifiers, and feed additives.

[0003] In Corynebacterium glutamicum, the production of threonine from oxaloacetate requires five catalytic steps, namely aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), homoserine dehydrogenase (encoded by hom), homoserine kinase (thrB), and threonine synthase (encoded by thrC). Hermann Sahm et al. have been dedicated to the development of Corynebacterium glutamicum strains with high threonine production and have made some breakthroughs, obtaining the feedback-resistant hom gene (Reinscheid DJ, Eikmanns BJ, Sahm H. Analysis of a Corynebacterium glutamicum hom gene coding for afeedback-resistant homoserine dehydrogenase.[J]. Journal of Bacteriology, 1991, 173(10):3228-3230) and the lysC gene (Eikmanns BJ, Eggeling L, Sahm H. Molecular aspects of lysine, threonine, and isoleucine biosynthesis in Corynebacterium glutamicum.[J]. Antonie Van Leeuwenhoek, 1993, 64(2):145-163). Following Hermann Sahm, Lothar Eggling further explored this field by weakening the glyA gene encoding the threonine utilization pathway and overexpressing the threonine exporter ThrE, thereby increasing the threonine production from 49 mM to 67 mM (Simic P,Willuhn J,Sahm H, et al. Identification of glyA (Encoding SerineHydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2002, 68(7):3321-3327).

[0004] Current reports on the production of threonine using Corynebacterium glutamicum mainly focus on its synthetic pathway, with fewer reports on the TCA cycle and central metabolism. Furthermore, existing reports only provide preliminary studies on the threonine synthetic pathway and do not form a systematic framework. Summary of the Invention

[0005] The purpose of this invention is to enhance the ability of a strain to produce threonine by inactivating quinone pyruvate dehydrogenase, thereby providing a method for constructing a threonine-producing (L-threonine) strain.

[0006] To achieve the objectives of this invention, in a first aspect, the present invention provides a modified Corynebacterium microorganism, wherein the activity of pyruvate quinone dehydrogenase is reduced or lost compared to the unmodified microorganism, and the microorganism has an enhanced threonine production capacity compared to the unmodified microorganism. Preferably, the pyruvate quinone dehydrogenase has the NCBI reference sequence number WP_011015247.1, or an amino acid sequence with 90% similarity to it.

[0007] Furthermore, the reduction or loss of pyruvate quinone dehydrogenase activity in the microorganism is achieved by reducing the expression of the gene encoding pyruvate quinone dehydrogenase or knocking out the endogenous gene encoding pyruvate quinone dehydrogenase.

[0008] Methods such as mutagenesis, site-directed mutagenesis, or homologous recombination can be used to reduce the expression of the gene encoding pyruvate quinone dehydrogenase or to knock out the endogenous gene encoding pyruvate quinone dehydrogenase.

[0009] Furthermore, compared with unmodified microorganisms, the activity of enzymes related to the threonine synthesis pathway in the microorganisms is enhanced;

[0010] The enzymes associated with the threonine synthesis pathway are selected from at least one of aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase; preferably, their reference sequence numbers on NCBI are WP_003855724.1, WP_003854900.1, WP_011013506.1, WP_011013497.1, WP_011014183.1, and WP_011014964.1, or amino acid sequences with 90% similarity to the above reference sequences.

[0011] Preferably, the microorganism is any one of the following ① to ⑤:

[0012] ① Microorganisms with reduced or lost pyruvate quinone dehydrogenase activity and enhanced aspartate kinase and / or homoserine dehydrogenase activity;

[0013] ② Microorganisms with reduced or lost pyruvate quinone dehydrogenase activity and enhanced aspartate kinase, homoserine dehydrogenase and / or aspartate aminotransferase activity;

[0014] ③ Microorganisms with reduced or lost pyruvate quinone dehydrogenase activity and enhanced aspartate kinase, homoserine dehydrogenase and / or aspartate semialdehyde dehydrogenase activity;

[0015] ④ Microorganisms with reduced or lost pyruvate quinone dehydrogenase activity and enhanced aspartate kinase, homoserine dehydrogenase and / or homoserine kinase activity;

[0016] ⑤ Microorganisms with reduced or lost pyruvate quinone dehydrogenase activity and enhanced aspartate kinase, homoserine dehydrogenase and / or threonine synthase activity.

[0017] The enhancement of the activity of enzymes related to the threonine synthesis pathway in the microorganism is achieved by selecting from 1) to 6) below, or an optional combination thereof:

[0018] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;

[0019] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;

[0020] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

[0021] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;

[0022] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme;

[0023] 6) Enhancement is achieved by altering the nucleotide sequence encoding the enzyme.

[0024] Preferably, the Corynebacterium mentioned in this invention is Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), and more preferably Corynebacterium glutamicum ATCC 13032.

[0025] Secondly, the present invention provides a method for constructing a threonine-producing strain, the method comprising:

[0026] A. Attenuating the gene encoding pyruvate quinone dehydrogenase in Corynebacterium species capable of amino acid production to obtain gene-attenuated strains; the attenuation includes knocking out or reducing the expression of the pyruvate quinone dehydrogenase encoding gene; and / or

[0027] B. Enhance the enzymes related to the threonine synthesis pathway in the strain weakened by gene A in step A to obtain strains with enhanced enzyme activity.

[0028] The enhancement method is selected from the following 1) to 5), or any combination thereof:

[0029] 1) Enhancement is achieved by introducing a plasmid containing the gene encoding the enzyme;

[0030] 2) Enhanced by increasing the copy number of the gene encoding the enzyme on the chromosome;

[0031] 3) Enhancement is achieved by altering the promoter sequence of the gene encoding the enzyme on the chromosome;

[0032] 4) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme;

[0033] 5) Enhancement is achieved by altering the amino acid sequence of the enzyme.

[0034] Thirdly, the present invention provides a method for producing threonine, the method comprising the following steps:

[0035] a) Culturing the modified Corynebacterium genus to obtain a culture of the microorganism;

[0036] b) Collect the resulting threonine from the culture obtained in step a).

[0037] Fourthly, the present invention provides the application of knocking out or reducing the expression of the gene encoding pyruvate quinone dehydrogenase in threonine fermentation production or increasing threonine fermentation yield.

[0038] Furthermore, the fermentation yield of threonine was increased by inactivating quinone pyruvate dehydrogenase in Corynebacterium, a bacterium with amino acid production capabilities.

[0039] Preferably, the Corynebacterium mentioned in this invention is Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum phylogenetic tree https: / / www.ncbi.nlm.nih.gov / genome / 469), and more preferably Corynebacterium glutamicum ATCC 13032.

[0040] Fifthly, the present invention provides the application of the modified Corynebacterium genus microorganism or the threonine-producing strain constructed according to the above method in the production of threonine by fermentation or in increasing the yield of threonine by fermentation.

[0041] The aforementioned methods for modifying the strains, including gene enhancement and weakening, are all modification techniques known to those skilled in the art. See Man Zaiwei, Systematic Pathway Engineering of High-Yielding L-Arginine Corynebacterium [D]. Jiangnan University, 2016; Cui Yi, Metabolic Engineering of Corynebacterium Glutamate for L-Leucine Production [D]. Tianjin University of Science and Technology; Xu Guodong, Construction of L-Isoleucine-Producing Strains and Optimization of Fermentation Conditions. Tianjin University of Science and Technology, 2015.

[0042] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0043] This invention applies pyruvate quinone dehydrogenase-inactivated strains (Corynebacterium, such as Corynebacterium glutamicum) to threonine production, increasing threonine yield by up to 25%–45.8% compared to unmodified strains. Further combination with expression enhancement of at least one of the following in the threonine synthesis pathway—aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase—results in a 10–21 fold increase in threonine yield. This provides a new pathway for large-scale threonine production and has high application value. Detailed Implementation

[0044] This invention focuses on the effect of quinone pyruvate dehydrogenase inactivation on threonine production. Based on the *Corynebacterium glutamicum* ATCC 13032 strain, the encoding gene of quinone pyruvate dehydrogenase was inactivated, resulting in strain SMCT322 with a threonine content of 0.2 g / L. This demonstrates that the loss of quinone pyruvate dehydrogenase activity is beneficial to threonine production.

[0045] Building upon this, to further verify the impact of quinone pyruvate dehydrogenase activity loss on threonine production, the threonine synthesis pathway of the strains was further enhanced, mainly involving the enhancement or deregulation of at least one of the following: aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase. The shake-flask results showed that all threonine-producing strains exhibited increased threonine production capacity after the loss of quinone pyruvate dehydrogenase activity. Furthermore, compared to strains that only inactivated the gene encoding quinone pyruvate dehydrogenase, the strain combining the loss of quinone pyruvate dehydrogenase activity with the enhancement of enzyme expression in the threonine synthesis pathway demonstrated a greater advantage in threonine production.

[0046] Inactivation or weakening during the modification process includes promoter replacement, alteration of ribosome binding sites, point mutation, and sequence deletion. Expression enhancement during the modification process includes promoter replacement, alteration of ribosome binding sites, increase of copy number, and plasmid overexpression. All of these methods are well-known to researchers in the field. The above methods cannot be exhaustively described by example; in this specific embodiment, promoter enhancement is used as a representative example.

[0047] The present invention adopts the following technical solution:

[0048] One of the technical solutions of the present invention provides a method for producing threonine using a strain that has lost pyruvate quinone dehydrogenase activity.

[0049] The second technical solution of the present invention provides a method for producing threonine using a strain that has been inactivated by pyruvate quinone dehydrogenase and whose expression of at least one of the following enzymes has been enhanced or deregulated: aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, homoserine kinase, or threonine synthase.

[0050] The third technical solution of the present invention provides a method for producing threonine using a strain with inactivated pyruvate quinone dehydrogenase and enhanced expression of aspartate kinase and homoserine dehydrogenase.

[0051] The fourth technical solution of the present invention provides a method for producing threonine using a strain with enhanced expression of pyruvate quinone dehydrogenase and aspartate kinase, homoserine dehydrogenase, and aspartate aminotransferase.

[0052] The fifth technical solution of the present invention provides a method for producing threonine using a strain with inactivated pyruvate quinone dehydrogenase and enhanced expression of aspartate kinase, homoserine dehydrogenase, and aspartate semialdehyde dehydrogenase.

[0053] The sixth technical solution of the present invention provides a method for producing threonine using a strain with inactivated pyruvate quinone dehydrogenase and enhanced expression of aspartate kinase, homoserine dehydrogenase, and homoserine kinase.

[0054] The seventh technical solution of the present invention provides a method for producing threonine using a strain with inactivated pyruvate quinone dehydrogenase and enhanced expression of aspartate kinase, homoserine dehydrogenase, and threonine synthase.

[0055] The above-mentioned strains are Corynebacterium, preferably Corynebacterium glutamicum, and most preferably Corynebacterium glutamicum ATCC 13032.

[0056] The gene encoding the protein involved in this invention is as follows:

[0057] Pyruvate quinone dehydrogenase, encoded by the gene poxB / pqo, NCBI IDs: cg2891, cgl2610, NCgl2521.

[0058] Aspartate kinase, encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.

[0059] Aspartate semialdehyde dehydrogenase, the encoding gene name is asd, NCBI number: Cgl0252, Cg0307, ​​NCgl0248.

[0060] Homoserine dehydrogenase, encoded by gene name hom, NCBI ID: Cg1337, Cgl1183, NCgl1136.

[0061] Threonine synthase, encoded by gene name thrC, NCBI ID: cg2437, Cgl2220, NCgl2139.

[0062] Homoserine kinase, encoding gene thrB, NCBI ID: cg1338, Cgl1184, NCgl1137.

[0063] Aspartate aminotransferase, encoding gene aspB, NCBI ID: cg0294, Cgl0240, NCgl0237.

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0065] The experimental materials used in the following examples are as follows:

[0066]

[0067]

[0068] The experimental methods involved in the following embodiments are as follows:

[0069] The PCR amplification system is as follows:

[0070] Sterilized deionized water 29 5×pfu buffer 10 2.5mM dNTP 5 10μM upstream primer 2 10μM downstream primer 2 Pfu 1 template 1 (Add up to 2 μL of fusion PCR template) total 50

[0071] The PCR amplification procedure is as follows:

[0072]

[0073] Strain modification methods:

[0074] 1. Seamless assembly reaction procedure: Refer to the ClonExpress MultiS One Step Cloning Kit instruction manual.

[0075] 2. Conversion method: Refer to the Trans1-T1 Phage Resistant Chemically Competent Cell instruction manual.

[0076] 3. Preparation of competent cells: Refer to C. glutamicum Handbook, Chapter 23.

[0077] Example 1: Construction of a genome-modified plasmid for a strain

[0078] 1. Construction of recombinant plasmid pK18mobsacB-ΔpoxB using a pyruvate quinone dehydrogenase inactivation protocol.

[0079] Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification with the poxB1 / poxB2 primer pair yielded the upstream homologous arm 'up', and PCR amplification with the poxB3 / poxB4 primer pair yielded the downstream homologous arm 'dn'. Fusion PCR with the poxB1 / poxB4 primer pair using 'up' and 'dn' as templates yielded the full-length fragment ΔpoxB. pK18mobsacB was digested with BamHI / HindIII. Both fragments were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-ΔpoxB.

[0080] 2. Aspartate kinase expression enhancement protocol: recombinant plasmid pK18mobsacB-Psod-lysC g1a-T311I Construction

[0081] Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification was performed using primers P21 / P22 to obtain the upstream homologous arm *up*, primers P23 / P24 to obtain the promoter fragment *Psod*, primers P25 / P26 to obtain *lysCg1a-T311I*, and primers P27 / P28 to obtain the downstream homologous arm *dn*. Fusion PCR was performed using primers P21 / P24 with *up* and *Psod* as templates to obtain the fragment *up-Psod*. Fusion PCR was performed using primers P21 / P28 with *up-Psod*, *lysCg1a-T311I*, and *dn* as templates to obtain the full-length fragment *up-Psod-lysCg1a-T311I-dn*. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P was obtained. sod -lysC g1a-T311I .

[0082] Wherein, g1a indicates that the first base of the start codon of the lysC gene (the wild-type lysC gene sequence is shown in SEQ ID NO:1) is mutated from g to a, and T311I indicates that the 311th amino acid of the aspartate kinase encoded by the lysC gene is mutated from T to I.

[0083] 3. Homoserine dehydrogenase expression enhancement protocol using recombinant plasmid pK18mobsacB-P cspB -hom G378E Construction

[0084] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification with primers P29 / P30 yielded the upstream homologous arm up. Using the genome of ATCC14067 as a template, PCR amplification with primers P31 / P32 yielded the promoter fragment PcspB. Using the genome of ATCC13032 as a template, PCR amplification with primers P33 / P34 yielded the homologous arm up. G378E PCR amplification was performed using primers P35 / P36 to obtain the downstream homologous arm dn. Fusion PCR was then performed using primers P29 / P32 with up and PcspB as templates to obtain the up-PcspB fragment. Finally, PCR amplification was performed using primers P29 / P36 with up-PcspB and hom... G378E Using dn as a template, fusion PCR was performed to obtain the full-length fragment up-PcspB-hom. G378E-dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P. cspB -hom G378E .

[0085] 4. Aspartate semialdehyde dehydrogenase expression enhancement protocol: recombinant plasmid pK18mobsacB-P sod -asd construction

[0086] Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification with primers P1 / P2 yielded the upstream homologous arm up, PCR amplification with primers P3 / P4 yielded the promoter fragment Psod, and PCR amplification with primers P5 / P6 yielded the downstream homologous arm dn. Fusion PCR using primers P1 / P6 with up, Psod, and dn as templates yielded the full-length fragment up-Psod-dn. pK18mobsacB was digested with BamHI / HindIII. Both fragments were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P. sod -asd.

[0087] 5. Aspartate aminotransferase expression enhancement protocol using recombinant plasmid pK18mobsacB-P sod -aspB construction

[0088] Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification with primers P103 / P104 yielded the upstream homologous arm up; PCR amplification with primers P105 / P106 yielded the promoter fragment Psod; and PCR amplification with primers P107 / P108 yielded the downstream homologous arm dn. Fusion PCR using primers P103 / P108 with up, Psod, and dn as templates yielded the full-length fragment up-Psod-dn. pK18mobsacB was digested with BamHI / HindIII. Both fragments were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P. sod -aspB.

[0089] 6. Homoserine kinase expression enhancement protocol using recombinant plasmid pK18-P cspB -thrB construction

[0090] Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, PCR amplification with primers P7 / P8 yielded the upstream homologous arm *up*. Using the genome of ATCC14067 as a template, PCR amplification with primers P9 / P10 yielded the promoter fragment *PcspB*. Using the genome of ATCC13032 as a template, PCR amplification with primers P11 / P12 yielded the downstream homologous arm *dn*. Fusion PCR using primers P7 / P12 with *up*, *PcspB*, and *dn* as templates yielded the full-length fragment *up-PcspB-dn*. pK18mobsacB was digested with BamHI / HindIII. Both fragments were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18-P. cspB -thrB.

[0091] 7. Threonine synthase expression enhancement plasmid pK18mobsacB-P sod -thrC g1a Construction

[0092] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification with primers P37 / P38 yielded the upstream homologous arm up, and PCR amplification with primers P39 / P40 yielded the promoter fragment Psod-thrC. g1a PCR amplification was performed using primers P41 / P42 to obtain the downstream homologous arm dn. Primers P37 / P42 were used with up and Psod-thrC. g1a Using dn as a template, fusion PCR was performed to obtain the full-length fragment up-Psod-thrC. g1a -dn. pK18mobsacB was digested with BamHI / HindIII. Both were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P. sod -thrC g1a .

[0093] Wherein, g1a indicates that the first base of the start codon of the thrC gene (the wild-type thrC gene sequence is shown in SEQ ID NO:2) is mutated from g to a. The primers used in the construction process are shown in Table 1:

[0094] Table 1

[0095]

[0096]

[0097] Note: Bold text and underlined text indicate primers that introduce the corresponding point mutations.

[0098] Example 2 Construction of Genome-Modified Strains

[0099] 1. Construction of pyruvate quinone dehydrogenase-inactivated strains

[0100] ATCC13032 competent cells were prepared according to the classic C. glutamicum Handbook (Chart 23). The recombinant plasmid pK18mobsacB-ΔpoxB was transformed into these competent cells by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin. The genes of interest were inserted into the chromosome due to homology. The selected transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (10⁻⁶ m / s). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT322.

[0101] 2. Enhanced expression of aspartate kinase and construction of deregulated strains

[0102] ATCC13032 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18mobsacB-Psod-lysC was used. g1a-T311I Competent cells were transformed by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT323.

[0103] 3. Construction of strains for enhancing homoserine dehydrogenase expression

[0104] SMCT323 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18mobsacB-P was used. cspB -hom G378E Competent cells were transformed by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT324.

[0105] 4. Construction of strains with enhanced expression of aspartate semialdehyde dehydrogenase

[0106] SMCT324 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18mobsacB-P was used. sod -asd cells were transformed into competent cells using electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT325.

[0107] 5. Construction of strains with enhanced aspartate aminotransferase expression

[0108] SMCT324 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18mobsacB-P was used. sod -aspB was used to transform competent cells using electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m / s). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT326.

[0109] 6. Construction of strains with enhanced expression of homoserine kinase

[0110] SMCT324 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18-P was used. cspB -thrB was used to transform competent cells via electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT327.

[0111] 7. Construction of strains with enhanced threonine synthase expression

[0112] SMCT324 competent cells were prepared according to the classic C. glutamicum Handbook (Chartter 23). Recombinant plasmid pK18mobsacB-P was used.sod -thrC g1a Competent cells were transformed by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin, where the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genome. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strain, named SMCT328.

[0113] 8. Construction of pyruvate quinone dehydrogenase-inactivated strains

[0114] SMCT324, SMCT325, SMCT326, SMCT327, and SMCT328 competent cells were prepared according to the classic C. glutamicum Handbook (Chart 23). The recombinant plasmid pK18mobsacB-ΔpoxB was transformed into these competent cells by electroporation, and transformants were screened on selective medium containing 15 mg / L kanamycin. The genes of interest were inserted into the chromosome due to homology. The screened transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, a second recombination occurred in the transformants, removing the vector sequence from the genome through gene exchange. The culture was serially diluted (10⁻⁶ m / s). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. The strains growing on the sucrose medium did not carry the inserted vector sequence in their genomes. The target sequence was amplified by PCR, and nucleotide sequencing analysis yielded the target mutant strains, named SMCT329, SMCT330, SMCT331, SMCT332, and SMCT333. The obtained strains are shown in Table 2.

[0115] Table 2

[0116] SMCT322 ATCC13032,ΔpoxB SMCT323 <![CDATA[ATCC13032,P sod -C court g1a-T311I ]]> SMCT324 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ]]> SMCT325 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -asd]]> SMCT326 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -aspB]]> SMCT327 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -home G378E ,P cspB -thrB]]> SMCT328 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -thrC g1a ]]> SMCT329 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,ΔpoxB]]> SMCT330 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -asd,ΔpoxB]]> SMCT331 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -aspB,ΔpoxB]]> SMCT332 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P cspB -thrB,ΔpoxB]]> SMCT333 <![CDATA[ATCC13032,P sod -lysC g1a-T311I ,P cspB -hom G378E ,P sod -thrC g1a ,ΔpoxB]]>

[0117] Example 3: Construction of strain for shake-flask verification

[0118] 1. Culture medium

[0119] Seed activation medium: BHI 3.7%, agar 2%, pH 7.

[0120] Seed culture medium: peptone 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, ammonium sulfate 16 g / L, urea 8 g / L, potassium dihydrogen phosphate 10.4 g / L, dipotassium hydrogen phosphate 21.4 g / L, biotin 5 mg / L, magnesium sulfate 3 g / L, glucose 50 g / L, pH 7.2.

[0121] Fermentation medium: corn steep liquor 50 mL / L, glucose 30 g / L, ammonium sulfate 4 g / L, MOPS 30 g / L, potassium dihydrogen phosphate 10 g / L, urea 20 g / L, biotin 10 mg / L, magnesium sulfate 6 g / L, ferrous sulfate 1 g / L, vitamin B1·HCl 40 mg / L, calcium pantothenate 50 mg / L, nicotinamide 40 mg / L, manganese sulfate 1 g / L, zinc sulfate 20 mg / L, copper sulfate 20 mg / L, pH 7.2.

[0122] 2. Production of L-threonine by shake-flask fermentation with engineered bacteria

[0123] (1) Seed culture: SMCT181, SMCT182, SMCT183, SMCT184, SMCT185, SMCT186, SMCT187, SMCT188, SMCT189, SMCT190, SMCT191, SMCT192, SMCT193, SMCT194, SMCT195, SMCT196 and SMCT197 slant seeds were looped into 500 mL Erlenmeyer flasks containing 20 mL of seed culture medium and cultured at 30 °C and 220 r / min for 16 h with shaking.

[0124] (2) Fermentation culture: 2 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation culture medium and cultured at 33 °C and 220 r / min for 24 h.

[0125] (3) Take 1 mL of fermentation broth and centrifuge (12000 rpm, 2 min), collect the supernatant, and use HPLC to detect L-threonine in the fermentation broth of engineered bacteria and control bacteria.

[0126] The results of threonine shake-flask fermentation are shown in Table 3:

[0127] Table 3

[0128] ATCC13032 25 — SMCT322 25 0.2 SMCT324 23 2.4 SMCT329 23 3.5 SMCT325 23 2.8 SMCT330 23 3.8 SMCT326 24 3.0 SMCT331 24 4.1 SMCT327 24 3.2 SMCT332 24 4.0 SMCT328 23 3.0 SMCT333 23 4.2

[0129] Table 3 shows that the threonine production of strains after pyruvate quinone dehydrogenase inactivation was increased compared to before inactivation, with the increase ranging from 20.5% to 45.8%, and the highest threonine production reaching 4.2 g / L. The threonine production varied among different pyruvate quinone dehydrogenase-inactivated strains, ranging from 0.2 g / L to 1.2 g / L, indicating that the inactivation of pyruvate quinone dehydrogenase has different effects depending on the combination with different sites. Furthermore, when combined with an enhancement phase for at least one of the following expression pathways in threonine synthesis—aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, or threonine synthase—the threonine production increased by 10-21 times.

[0130] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> Construction method of threonine-producing strains <130> KHP211124123.7 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1266 <212> DNA <213> Corynebacterium glutamicum <400> 1 gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60 aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120 tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180 ccgccagctc gtgaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240 gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300 ggtgtgctca ccaccgagcg ccacgggaaac gcacgcattg ttgatgtcac tccaggtcgt 360 gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420 aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480 ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540 accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600 atgctgggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660 cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720 attgccggct ctatggagga tattcctgtg gaaagcag tccttaccgg tgtcgcaacc 780 gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840 aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900 tcttctgtag aagacggcac caccgacatc accttcacct gccctcgttc cgacggccgc 960 cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020 gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt 1080 accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc 1140 tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca 1200 ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga 1260 cgctaa 1266 <210> 2 <211> 1446 <212> DNA <213> Corynebacterium glutamicum <400> 2 gtggactaca tttcgacgcg tgatgccagc cgtacccctg cccgcttcag tgatattttg 60 ctgggcggtc tagcaccaga cggcggcctg tacctgcctg caacctaccc tcaactagat 120 gatgcccagc tgagtaaatg gcgtgaggta ttagccaacg aaggatacgc agctttggct 180 gctgaagtta tctccctgtt tgttgatgac atcccagtag aagacatcaa ggcgatcacc 240 gcacgcgcct acacctaccc gaagttcaac agcgaagaca tcgttcctgt caccgaactc 300 gaggacaaca tttacctggg ccacctttcc gaaggcccaa ccgctgcatt caaagacatg 360 gccatgcagc tgctcggcga acttttcgaa tacgagcttc gccgccgcaa cgaaaccatc 420 aacatcctgg gcgctacctc tggcgatacc ggctcctctg cggaatacgc catgcgcggc 480 cgcgagggaa tccgcgtatt catgctgacc ccagctggcc gcatgacccc attccagcaa 540 gcacagatgt ttggccttga cgatccaaac atcttcaaca tcgccctcga cggcgttttc 600 gacgattgcc aagacgtagt caaggctgtc tccgccgacg cagaattcaa aaaagacaac 660 cgcatcggtg ccgtgaactc catcaactgg gcacgcctta tggcacaggt tgtgtactac 720 gtttcctcat ggatccgcac cacaaccagc aatgaccaaa aggtcagctt ctccgtacca 780 accggcaact tcggtgacat ttgcgcaggc cacatcgccc gccaaatggg acttcccatc 840 gatcgcctca tcgtggccac caacgaaaac gatgtgctcg acgagttctt ccgtaccggc 900 gactaccgag tccgcagctc cgcagacacc cacgagacct cctcaccttc gatggatatc 960 tcccgcgcct ccaacttcga gcgtttcatc ttcgacctgc tcggccgcga cgccacccgc 1020 gtcaacgatc tatttggtac ccaggttcgc caaggcggat tctcactggc tgatgacgcc 1080 aactttgaga aggctgcagc agaatacggt ttcgcctccg gacgatccac ccatgctgac 1140 cgtgtggcaa ccatcgctga cgtgcattcc cgcctcgacg tactaatcga tccccacacc 1200 gccgacggcg ttcacgtggc acgccagtgg aggacgagg tcaacacccc aatcatcgtc 1260 ctagaaactg cactcccagt gaaatttgcc gacaccatcg tcgaagcaat tggtgaagca 1320 cctcaaactc cagagcgttt cgccgcgatc atggatgctc cattcaaggt ttccgaccta 1380 ccaaacgaca ccgatgcagt taagcagtac atagtcgatg cgattgcaaa cacttccgtg 1440 aagtaa 1446

Claims

1. A modified Corynebacterium genus microorganism, characterized in that, Compared to unmodified microorganisms, the microorganisms lost the activity of pyruvate quinone dehydrogenase and had enhanced threonine production capacity compared to unmodified microorganisms. The loss of pyruvate quinone dehydrogenase activity in the microorganism was achieved by knocking out the endogenous gene encoding pyruvate quinone dehydrogenase through homologous recombination. Compared with unmodified microorganisms, the microorganisms exhibit enhanced activity of enzymes related to the threonine synthesis pathway within them. The enzymes related to the threonine synthesis pathway are selected from aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase. The microorganism is any one of the following ① to ⑤: ① Microorganisms that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase and homoserine dehydrogenase activity; ② Microorganisms that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and aspartate aminotransferase activities; ③ Microorganisms that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and aspartate semialdehyde dehydrogenase activities. ④ Microorganisms that have lost pyruvate quinone dehydrogenase activity and have increased activity of aspartate kinase, homoserine dehydrogenase and homoserine kinase; ⑤ Microorganisms that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and threonine synthase activity; The microorganism is Corynebacterium glutamicum (Bacillus glutamate). Corynebacterium glutamicum ATCC 13032.

2. The microorganism according to claim 1, characterized in that, The enhancement of the activity of enzymes related to the threonine synthesis pathway in the microorganism is selected from the following 1) to 3): 1) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme; 2) Enhancement is achieved by altering the amino acid sequence of the enzyme; specifically, threonine at position 311 of aspartate kinase is mutated to isoleucine, and glycine at position 378 of homoserine dehydrogenase is mutated to glutamic acid. 3) Enhancement is achieved by altering the nucleotide sequence of the encoded enzyme; specifically, the first base of the start codon of the lysC gene is mutated from g to a, the gene encoding aspartate kinase is mutated to a mutant gene in which the threonine at position 311 of the encoded aspartate kinase is mutated to isoleucine, and the gene encoding homoserine dehydrogenase is mutated to a mutant gene in which the glycine at position 378 of the encoded homoserine dehydrogenase is mutated to glutamate.

3. A method for constructing a threonine-producing strain, characterized in that, The method includes: A. Attenuating the gene encoding pyruvate quinone dehydrogenase in Corynebacterium species capable of amino acid production to obtain gene-attenuated strains; wherein attenuation is performed by knocking out the gene encoding pyruvate quinone dehydrogenase; and B. Enhance the enzymes related to the threonine synthesis pathway in the strain weakened by gene A in step A to obtain strains with enhanced enzyme activity. The enhancement pathway is selected from the following 1)~2): 1) Enhancement is achieved by operatively linking a strong promoter to the gene encoding the enzyme; 2) Enhancement is achieved by altering the amino acid sequence of the enzyme; specifically, threonine at position 311 of aspartate kinase is mutated to isoleucine, and glycine at position 378 of homoserine dehydrogenase is mutated to glutamic acid. The enzymes related to the threonine synthesis pathway are selected from aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase. The strain is any one of the following ① to ⑤: ① Strains that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase and homoserine dehydrogenase activity; ② Strains that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and aspartate aminotransferase activities. ③ Strains that lose pyruvate quinone dehydrogenase activity and have enhanced aspartate kinase, homoserine dehydrogenase and aspartate semialdehyde dehydrogenase activities. ④ Strains that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and homoserine kinase activity; ⑤ Strains that have lost pyruvate quinone dehydrogenase activity and have increased aspartate kinase, homoserine dehydrogenase and threonine synthase activity. The Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032.

4. A method for producing threonine, characterized in that, The method includes the following steps: a) Cultivate the microorganism according to claim 1 or 2 to obtain a culture of the microorganism; b) Collect the resulting threonine from the culture obtained in step a).

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